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Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes.

The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.

Amino Acid Sequence↗

[Phage sensitivity of Streptococcus group A strains isolated in glomerulonephritis].

The study of phage-sensitive strains of group A streptococci of different serological types, isolated from glomerulonephritis patients in Czechoslovakia, has confirmed the data previously obtained in Moscow. As revealed in this study, all strains of type 12, containing M-antigen, are sensitive to group 1 phage from the phage collection used in the investigation. Some of the strains of type 12, containing M-antigen and isolated from healthy carriers, have also proved to be sensitive to group 1 phages. Thus, streptococcal strains of type 12, containing M-substance and found to be the main causative agents of glomerulonephritis, have shown similar phage sensitivity when isolated in different geographical zones. For this reason, group 1 phages can be used for the detection of some "nephritogenic" strains among the population.

Acute Disease↗

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage.

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types-those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.

Journal Article↗

Biotyping, serotyping and phage typing of Streptococcus faecalis isolated from dental plaque in the human mouth.

Thirty Streptococcus faecalis isolates from mixed dental plaque samples were classified into four groups on the basis of biotype, tetracycline susceptibility, phage type and serotype combinations. The organisms were from patients on haemodialysis, from staff of the dialysis unit, and from controls. Three biotypes were distinguished by seven biochemical tests: production of acid from inositol, sucrose and xylose; rapid or delayed production of acid from sorbitol; gelatin liquefaction; and production of alkaline phosphatase and beta-galactosidase. With a set of eight typing antisera for S. faecalis, 15 strains were non-typable, 12 were serotype 1 and three were serotype 19. With a set of 17 bacteriophages specific for S. faecalis, all of the oral isolates were typable; 40% were lysotype I1 and the remainder lysotype V6b. On the basis of biotype-serotype-phage-type combinations, indications of possible spread of strains between haemodialysis patients and dialysis unit staff were obtained. Biotyping and serotyping of 13 German isolates of S. faecalis of phage type I1 from four clinical sources and tripartite typing of three control strains provided additional evidence for the potential of biotyping in distinguishing between strains of identical serotype and phage type. One oral isolate of S. faecium was of phage type XX. None of the oral isolates of S. faecalis, of which 14 exhibited delayed sorbitol fermentation, reacted with group-G streptococcal grouping reagents or antiserum. Slow sorbitol fermentation does not appear to be a definitive phenotypic marker for S. faecalis strains possessing antigens that react with both group-D and group-G grouping reagents.

Adolescent↗

Transduction of drug resistance to tetracycline, chloramphenicol, macrolides, lincomycin and clindamycin with phages induced from Streptococcus pyogenes.

Strains of Streptococcus pyogenes isolated from pediatric patients with acute infections which were resistant to one or more of the antibiotics, tetracycline (TC), chloramphenicol (CP), macrolide antibiotics (erythromycin, kitasamycin, oleandomycin, josamycin), lincomycin (LCM) and clindamycin (CLM), were used for transduction of drug resistance. These drug-resistant strains were treated with mitomycin C to induce phages and transduction of drug resistance was attempted by means of phages so induced. It was found that transduction of resistance to the above antibiotics was possible. The transductants obtained on TC-containing selective agar plate were resistant to TC alone while those produced on CP- or erythromycin (EM)-containing selective agar plate were resistant to CP, macrolide antibiotics (Mac), LCM and CLM. From this finding, it was inferred that transduction of resistance to TC, CP, Mac, LCM and CLM via phages occurred in two different patterns, i.e., transfer of resistance to TC alone and that of resistance to CP, Mac, LCM and CLM. All of the transductants obtained were found to belong to group A. In T-typing, they were of the same T-12 type as the donor and recipient strains in a majority of cases though some were not typable.

Anti-Bacterial Agents↗

Lytic action of cloned pneumococcal phage lysis genes in Streptococcus pneumoniae.

The genes hbl3, cpl1 and cpl7 coding for the pneumococcal phage lytic enzymes HBL3, CPL1 and CPL7, respectively, have been cloned into shuttle plasmids that can replicate in Streptococcus pneumoniae and Escherichia coli. All these genes were expressed in E. coli under the control of either the lytP promoter of the lytA gene, which codes for the major pneumococcal autolysin, or the promoter of the tetracycline-resistance gene (tetP). In contrast, cpl1 and cpl7 genes that code for lysozymes were expressed in pneumococcus only under the control of tetP, whereas the hbl3 gene that codes for an amidase can be expressed using either promoter. The phage lysozymes or amidase expressed in S. pneumoniae M31, a mutant deleted in the lytA gene coding for short chains, were placed under physiological control since these transformed bacteria grew as normal 'diplo' cells during the exponential phase and underwent autolysis only after long incubation at 37 degrees C. The lysis genes appear to be expressed constitutively in the transformed pneumococci, since sharply defined lysis of these cultures could be induced prematurely during the exponential phase of growth by addition of sodium deoxycholate.

Amidohydrolases↗

Rapid selection of phage-resistant mutants in Streptococcus thermophilus by immunoselection and cell sorting.

Immunoselection and flow cytometry allowed the isolation from Streptococcus thermophilus strain Str31 of double mutants displaying resistance to the phage phi31 and good acid production. Strain Str31 is very sensitive to phage phi31. This phage-host system seemed therefore particularly suitable to test the validity of the selection method adopted in this study. Mutants were stable with respect to both characters. The isolation of the double mutants required 4 to 5 days. The approach does not involve genetic manipulations and can therefore be an alternative to genetic engineering when this technology cannot be applied.

Adsorption↗

Lysogeny of Streptococcus pneumoniae with MM1 phage: improved adherence and other phenotypic changes.

Pneumococcal prophages are extremely frequent, but no role in pathogenesis has so far been attributed to them. We isolated a variant of phage MM1, named MM1-1998, from a serotype 24 strain of Streptococcus pneumoniae. We created three isogenic strain pairs (serotypes 3, 4, and 24) that differed only by the lysogenic presence of the MM1-1998 phage and did a phenotypic comparison. Lysogeny led to improved adherence to inert surfaces and pharyngeal cells compared to that with the cured variants of the strains. We found that lysogeny with MM1-1998 coincided with a more transparent phenotype and phage curing with more opaque colonies in all strain pairs, and we discovered that transparency was associated with more successful and stable lysogeny. Since transparency alone was possibly responsible for the adherence difference, we further compared the TIGR4 lysogen with an equally transparent variant of TIGR4 in order to reassess the role of phage or transparency separately. The results revealed that improved adherence was independently associated with lysogeny with the MM1-1998 phage. Other phenotypic differences such as faster growth, increased autolysis, and decreased intracellular hemolytic activity were more likely due to transparency. By improving the adherence of pneumococci, this prophage may contribute to their fitness and possibly to their persistence in humans.

Animals↗

Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains.

Pal and Cpl-1, two purified bacteriophage lytic enzymes, were tested for their in vitro activity, alone and in combination, against several serotypes of Streptococcus pneumoniae, including penicillin-resistant strains. The enzymes demonstrated synergism in their ability to cleave the bacterial peptidoglycan and thus may be more efficient for the prevention and elimination of pneumococcal colonization.

Penicillin Resistance↗

Restriction and modification in group N streptococci: effect of heat on development of modified lytic bacteriophage.

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 x 10 for tr plaqued on 799 and 2.1 x 10 for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50 degrees C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.

Journal Article↗

Primary structure and functional analysis of the lysis genes of Lactobacillus gasseri bacteriophage phi adh.

The lysis genes of the Lactobacillus gasseri bacteriophage phi adh were isolated by complementation of a lambda Sam mutation in Escherichia coli. Nucleotide sequencing of a 1,735-bp DNA fragment revealed two adjacent coding regions of 342 bp (hol) and 951 bp (lys) in the same reading frame which appear to belong to a common transcriptional unit. Proteins corresponding to the predicted gene products, holin (12.9 kDa) and lysin (34.7 kDa), were identified by in vitro and in vivo expression of the cloned genes. The phi adh holin is a membrane-bound protein with structural similarity to lysis proteins of other phage, known to be required for the transit of murein hydrolases through the cytoplasmic membrane. The phi adh lysin shows homology with mureinolytic enzymes encoded by the Lactobacillus bulgaricus phage mv4, the Streptococcus pneumoniae phage Cp-1, Cp-7, and Cp-9, and the Lactococcus lactis phage phi LC3. Significant homology with the N termini of known muramidases suggests that phi adh lysin acts by a similar catalytic mechanism. In E. coli, the phi adh lysin seems to be associated with the total membrane fraction, from which it can be extracted with lauryl sarcosinate. Either one of the phi adh lysis proteins provoked lysis of E. coli when expressed along with holins or lysins of phage lambda or Bacillus subtilis phage phi 29. Concomitant expression of the combined holin and lysin functions of phi adh in E. coli, however, did not result in efficient cell lysis.

Amino Acid Sequence↗

Comparative genomics of lactococcal phages: insight from the complete genome sequence of Lactococcus lactis phage BK5-T.

Lactococcus lactis phage BK5-T and Streptococcus thermophilus phage Sfi21, two cos-site temperate Siphoviridae with 40-kb genomes, share an identical genome organization, sequence similarity at the amino acid level over about half of their genomes, and nucleotide sequence identity of 60% over the DNA packaging and head morphogenesis modules. Siphoviridae with similarly organized genomes and substantial protein sequence similarity were identified in several genera of low-GC-content Gram-positive bacteria. These phages demonstrated a gradient of relatedness ranging from nucleotide sequence similarity to protein sequence similarity to gene map similarity over the DNA packaging and head morphogenesis modules. Interestingly, the degree of relatedness was correlated with the evolutionary distance separating their bacterial hosts. These observations suggest elements of vertical evolution in phages. The structural genes from BK5-T shared no sequence relationships with corresponding genes/proteins from lactococcal phages belonging to distinct lactococcal phage species, including phage sk1 (phage species 936) that showed a closely related gene map. Despite a clearly distinct genome organization, lactococcal phages sk1 and c2 showed nine sequence-related proteins. Over the early gene cluster phage BK5-T shared nine regions of high nucleotide sequence similarity, covering at most two adjacent genes, with lactococcal phage r1t (phage species P335). Over the structural genes, the closest relatives of phage r1t were not lactococcal phages belonging to other phage species, but Siphoviridae from Mycobacteria (high-GC-content Gram-positive bacteria). Evidence for recent horizontal gene transfer between distinct phage species was obtained for dairy phages, but these transfers were limited to phages infecting the same bacterial host species.

Computational Biology↗

[Studies on the relationship of temperent phages and bacteriocines of streptococcus faecium (author's transl)].

Two bacteriocins (enterocin E1A and E1B) as well as a complete bacteriophage (PE1) were produced by Streptococcus faecium strain E1. Although the phage could be demonstrated by electron microscopy it was not possible to observe phages or phage-like particles in the purified preparation of the large enterocin E1B. Phage PE1 had a much smaller activity spectrum than that of enterocin E1A and E1B, inhibiting only one strain of Streptococcus faecium and one strain of Streptococcus salivarius. The enterocins were not neutralized by antiphage sera, thereby suggesting that the enterocins and the phage are chemically unrelated.

Animals↗

Characterization of a temperate Streptococcus thermophilus bacteriophage and its genetic relationship with lytic phages.

The temperate Streptococcus thermophilus bacteriophage phi SFi21 showed an 38-kb-long double-stranded DNA genome with cohesive ends. A single integration site was used in lysogens established in three different S. thermophilus strains. The attP and attB sites were localized on the restriction map of phage DNA and by hybridization on pulsed field separated bacterial DNA. All laboratory-established lysogens showed in addition to integrated prophage DNA unintegrated monomer phage DNA with unligated cos sites. The genetic relatedness of phi SFi21 DNA with DNA from lytic phages was studied in dot blot and Southern blot hybridization by using individual restriction fragments of phiSFi21 DNA as probes. Lytic group I phages hybridized with fragments of the central and the right part of the phiSFi21 genome but failed to hybridize with a fragment joining both parts. Lytic group II phages showed hybridization with the right half of the phiSFi21 genome. In lytic group IV phages, biologically a heterogeneous group, many different combinations of cross hybridization were detected in accordance with the hypothesis of the modular evolution of phage genomes.

Attachment Sites, Microbiological↗

[Selection of phage-resistent clones of streptococcus group A and their properties].

In studying the effects induced by virulent phage CAI in the sensitive cultures of streptococcus, group A, a possibility was shown of selection of phage-resistant clones with the altered enzymatic activity. These clones lost their capacity to produce proteinase and retained residual lipoproteinase activity. This evidence correlates with literature data indicating that phage-resistant streptococci served as good producers of M-protein--the main virulence factor. Infection of the culture producing streptokinase with phage CAI with a definite infection multiplicity led to an increase of the enzyme activity in the culture fluid. This process was accompanied by selection of the resistant strains characterized by greater streptokinase production and greater enzyme stability. As suggested, the latter could result from the absence of proteolytic activity in the phage-resistant clone.

Bacteriophages↗